PROJECT SUMMARY Inadequate stimulation of the neonatal immune system and the requirement for multiple booster administrations have limited the efficacy of the majority of current vaccine formulations. These issues contribute to a window of vulnerability during which neonates and infants are highly susceptible to infection, resulting in over 2 million deaths worldwide each year. Despite numerous shortcomings, including waning protection following childhood and ineffectiveness for adults, the Bacille Calmette-Guerin (BCG) attenuated vaccine against Mycobacterium tuberculosis (Mtb) safely elicits Th1 neonatal immune responses and requires only a single administration at the time of birth. Thus, although BCG is in dire need of improvement, it is not burdened by several critical deficiencies effecting most currently administered immunizations. Furthermore, patients immunized with BCG and other attenuated vaccines have demonstrated enhanced and more effective immune responses upon infection with unrelated pathogens later in life resulting lowered overall mortality. These off-target nonspecific benefits are often referred to as heterologous effects and suggest that immunostimulation by BCG and other attenuated vaccines may ?train? neonatal immune cells for lasting immunological advantages. Thus there is a critical need to improve the majority of current vaccine formulations to achieve one-shot neonatal immunization while ensuring programming of neonatal immunity for retention of heterologous effects of attenuated vaccines. Furthermore, the current global shortage in BCG calls attention to the current lack of scalable synthetic formulations that can achieve similar and potentially improved efficacy relative to immunization with live attenuated pathogens. By identifying and mechanistically investigating the principle immunological responses elicited by the BCG vaccine, synthetic formulations can be rationally designed to mimic the beneficial immunostimulatory pathways of BCG while avoiding and improving upon its shortcomings. Both the immunological pathways and duration of immunostimulation elicited by BCG vaccination have been linked to its efficacy as a neonatal vaccine. This proposal aims to apply a synthetic vaccine depot loaded with strategically selected adjuvant combinations and possessing highly tunable release kinetics to mimic key pathways and duration of immunostimulation of BCG. The influences of these pathways on leukocytes, particularly with respect to memory T cell generation and heterologous immunity, will be investigated in vivo with a humanized mouse model and in vitro using human derived immune cell populations. The following aims will be achieved: ? Aim 1: Synthetically mimic the release kinetics of BCG for sustained immunostimulation in an in vivo neonatal vaccination model. ? Aim 2: Identify the principle immunostimulatory and ?training? effects of the most and least effective formulations from Aim 1 on human neonatal leukocytes in vitro and TLR8 humanized mice in vivo.